Method for specific cleavage of C alpha-C bond and side chain of protein and peptide, and method for determining amino acid sequence

Abstract

The present invention provides a method for specifically cleaving a Cα-C bond of a peptide backbone and/or a side chain of a protein and a peptide, and a method for determining amino acid sequences of protein and peptide. A method for specifically cleaving a Cα-C bond of a peptide backbone and/or a side chain bond of a protein or a peptide, comprising irradiating a protein or a peptide with laser light in the presence of at least one hydroxynitrobenzoic acid selected from the group consisting of 3-hydroxy-2-nitrobenzoic acid, 4-hydroxy-3-nitrobenzoic acid, 5-hydroxy-2-nitrobenzoic acid, 3-hydroxy-5-nitrobenzoic acid, and 4-hydroxy-2-nitrobenzoic acid. A method for determining an amino acid sequence of a protein or a peptide, comprising irradiating a protein or a peptide with laser light in the presence of the above specific hydroxynitrobenzoic acid to specifically cleave a Cα-C bond of a peptide backbone and/or a side chain bond, and analyzing generated fragment ions by mass spectrometry.

What is claimed is: 
     
       1. A method for specifically cleaving a Cα-C bond of a peptide backbone and/or a side chain bond of a protein or a peptide, comprising irradiating a protein or a peptide with laser light in the presence of at least one hydroxynitrobenzoic acid selected from the group consisting of:
 3-hydroxy-2-nitrobenzoic acid: 
 
       
         
           
           
               
               
           
         
         4-hydroxy-3-nitrobenzoic acid: 
       
       
         
           
           
               
               
           
         
         5-hydroxy-2-nitrobenzoic acid: 
       
       
         
           
           
               
               
           
         
         3-hydroxy-5-nitrobenzoic acid: 
       
       
         
           
           
               
               
           
         
         and 
         4-hydroxy-2-nitrobenzoic acid: 
       
       
         
           
           
               
               
           
         
         to specifically cleave the Cα-C bond of the peptide backbone and/or the side chain bond, wherein the hydroxynitrobenzoic acid promotes the specific cleavage of the Cα-C bond of the peptide backbone and/or the side chain bond. 
       
     
     
       2. A method for determining an amino acid sequence of a protein or a peptide, comprising:
 irradiating a protein or a peptide with laser light in the presence of at least one hydroxynitrobenzoic acid selected from the group consisting of: 
 3-hydroxy-2-nitrobenzoic acid: 
 
       
         
           
           
               
               
           
         
         4-hydroxy-3-nitrobenzoic acid: 
       
       
         
           
           
               
               
           
         
         5-hydroxy-2-nitrobenzoic acid: 
       
       
         
           
           
               
               
           
         
         3-hydroxy-5-nitrobenzoic acid: 
       
       
         
           
           
               
               
           
         
         and 
         4-hydroxy-2-nitrobenzoic acid: 
       
       
         
           
           
               
               
           
         
         to specifically cleave a Cα-C bond of a peptide backbone and/or a side chain bond; and 
         analyzing generated fragment ions by mass spectrometry, 
         wherein the hydroxynitrobenzoic acid promotes the specific cleavage of the Cα-C bond of the peptide backbone and/or the side chain bond. 
       
     
     
       3. A method for determining an amino acid sequence of a protein or a peptide, comprising:
 irradiating a protein or a peptide with laser light in the presence of at least one hydroxynitrobenzoic acid selected from the group consisting of 3-hydroxy-2-nitrobenzoic acid, 4-hydroxy-3-nitrobenzoic acid, 5-hydroxy-2-nitrobenzoic acid, 3-hydroxy-5-nitrobenzoic acid, and 4-hydroxy-2-nitrobenzoic acid as a matrix to specifically cleave a Cα-C bond of a peptide backbone and/or a side chain bond; and 
 analyzing generated fragment ions by MALDI mass spectrometry, 
 wherein the hydroxynitrobenzoic acid promotes the specific cleavage of the Cα-C bond of the peptide backbone and/or the side chain bond. 
 
     
     
       4. The method for determining an amino acid sequence of a protein or a peptide according to  claim 2 , wherein as the generated fragment ions, a-series ion species and/or x-series ion species are analyzed. 
     
     
       5. The method for determining an amino acid sequence of a protein or a peptide according to  claim 2 , wherein as the generated fragment ions, a-series ion species and d-series ion species are analyzed.